Heterochromatin represents -30% of chromatin in flies and humans, is often found at centromeres and telomeres and remains condensed throughout the cell cycle. Heterochromatin is important for many cellular functions including gene silencing and accurate chromosomal association and segregation during the cell cycle and defects in heterochromatin can lead to developmental diseases and cancer. Using the yeast Saccharomyces cerevisiae as a model system our laboratory has contributed to the molecular understanding of the mechanism by which heterochromatin initiates, spreads along deacetylated histone H4 N termini and stops spreading through their acetylation. However, we have little understanding of the role of other histones or how their modification sites at the N termini or elsewhere are altered to allow the formation and maintenance of heterochromatin and accurate chromosomal association. The goal of this proposal is to use the sophisticated genetic, biochemical and microarray techniques of Saccharomyces cerevisiae to study the role of the H3 N terminus in the initiation and spreading of heterochromatin and to determine the factors with which the H3 N terminus interacts to help form heterochromatin structure. The mechanisms by which most sites in histones are deacetylated in heterochromatin and the acetyltransferase Sas2 acetylates histone H4 to block the spread of heterochromatin will be investigated. It has been discovered recently that H3-K56 is a novel site in the core of histone H3 that is essential for silencing in heterochromatin. The enzymes that regulate the acetylation state of H3-K56 and the molecular mechanism by which H3-K56 regulates silencing at telomeres and the silent mating locus HMR will be investigated. Finally, the chromatin factors that determine cohesion binding will be identified. These studies will expose conserved molecular mechanisms that control the formation of heterochromatin and promise to provide drug targets for the prevention of human disease. The histone proteins are the building blocks of chromosomes and provide surfaces for the interaction of proteins that regulate chromosomal structure and gene activity. Heterochromatin is a large condensed fraction of human chromosomes and is involved in silencing genes and enabling accurate transfer of chromosomes to dividing cells. These studies will investigate the molecular interactions between heterochromatin proteins and histones, interactions that promise to provide drug targets for the prevention of human diseases such as cancer.